(i) erbB Proto-oncogenes PA1 (ii) Neuregulins PA1 (iii) Neuregulin Domains PA1 (iv) Other Putative HER2 Activators PA1 (v) Biological Activities of Neuregulins
Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases are enzymes that catalyze this process. Receptor protein tyrosine kinases are believed to direct cellular growth via ligand-stimulated tyrosine phosphorylation of intracellular substrates. Growth factor receptor protein tyrosine kinases of the class I subfamily include the 170 kDa epidermal growth factor receptor (EGFR) encoded by the erbB gene. erbB has been causally implicated in human malignancy. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung and stomach. See Neal et al., Lancet, 1:366-368 (1985); Sainsbury et al., Lancet, 1:1389-1402 (1987); Yasui et al., Int. J. Cancer, 41: 211-217 (1988); and Veale et al., Cancer, 55:513-516 (1987).
The second member of the class I subfamily, p185.sup.neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The neu gene (also called erbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase. Amplification and/or overexpression of the human HER2 gene correlates with a poor prognosis in breast and ovarian cancers (Slamon et al., Science, 235:177-182 [1987]; and Slamon et al., Science, 244:707-712 [1989]). Overexpression of HER2 has also been correlated with other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder. See, among others, King et al., Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushigi et al., Mol Cell Biol., 6:955-958 (1986); Slamon et al. (1987), supra; Geurin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4: 81-88 (1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol, Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:354-357 (1990); and McCann et al., Cancer, 65: 88-92 (1990). Accordingly, Slamon et al. in U.S. Pat. No. 4,968,603 describe and claim various diagnostic assays for determining HER2 gene amplification or expression in tumor cells. Slamon et al. discovered that the presence of multiple gene copies of HER2 oncogene in tumor cells indicates that the disease is more likely to spread beyond the primary tumor site, and that the disease may therefore require more aggressive treatment than might otherwise be indicated by other diagnostic factors. Slamon et al. conclude that the HER2 gene amplification test, together with the determination of lymph node status, provides greatly improved prognostic utility.
Expression of the HER2 gene in non-cancerous tissue has also been investigated. For example, Cohen et al. found that the HER2 gene is expressed by Schwann cells during peripheral nerve development and wallerian degeneration. Accordingly, they concluded that p185.sup.HER2 plays a role in regulation of proliferation or differentiation of Schwann cells. Cohen et al., J. Neuroscience Res., 31:622-634 (1992).
A further related gene, called erbB3 or HER3, has also been described. See U.S. Pat. No. 5,183,884; Kraus et al., PNAS, U.S.A., 86: 9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus et al., PNAS, U.S.A., 90:2900-2904 (1993). Kraus et al. (1989) discovered that markedly elevated levels of erbB3 mRNA were present in certain human mammary tumor cell lines indicating that erbB3, like erbB and erbB2, may play a role in some human malignancies. Also, Kraus et al. (1993) showed that EGF-dependent activation of the erbB3 catalytic domain of a chimeric EGFR/erbB3 receptor resulted in a proliferative response in transfected NIH-3T3 cells. Furthermore, these researchers demonstrated that some human mammary tumor cell lines display a significant elevation of steady-state erbB3 tyrosine phosphorylation further indicating that this receptor may play a role in human malignancies. Accordingly, diagnostic bioassays utilizing nucleic acid encoding erbB3 are described by Kraus et al. in U.S. Pat. No. 5,183,884.
Recently, the class I subfamily of growth factor receptor protein tyrosine kinases was extended to include the HER4/p180.sup.erbB4 receptor. See EP Pat Appln No 599,274; Plowman et al., PNAS, U.S.A., 90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993). Plowman et al. found that increased HER4 expression closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Accordingly, diagnostic methods for detection of human neoplastic conditions (especially breast cancers) which evaluate HER4 expression are described in EP Pat Appln No. 599,274.
The quest for the activator of the HER2 oncogene has lead to the discovery of a family of polypeptides collectively called "neuregulins". These proteins appear to result from alternative splicing of a single gene which was mapped to the short arm of human chromosome 8 by Orr-Urtreger et al., PNAS, U.S.A., 90:1867-1871 (1993).
Holmes et al. isolated and cloned a family of polypeptide activators for the HER2 receptor which they termed heregulin-.alpha. (HRG-.alpha.), heregulin-.beta.1 (HRG-.beta.1), heregulin-.beta.2 (HRG-.beta.2), heregulin-.beta.2-like (HRG-.beta.2-like), and heregulin-.beta.3 (HRG-.beta.3). See Holmes et al., Science, 256:1205-1210 (1992); and WO 92/20798 as well as Hoffman et al., Science, 256:1129 (1992). The 45 kDa polypeptide, HRG-.alpha., was purified from the conditioned medium of the MDA-MB-231 human breast cancer cell line. These researchers demonstrated the ability of the purified heregulin polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF-7 breast tumor cells. Furthermore, the mitogenic activity of the heregulin polypeptides on SK-BR-3 cells (which express high levels of the HER2 receptor) was illustrated. Like other growth factors which belong to the EGF family, soluble HRG polypeptides appear to be derived from a membrane bound precursor (called pro-HRG) which is proteolytically processed to release the 45 kDa soluble form. These pro-HRGs lack a N-terminal signal peptide.
While heregulins are substantially identical in the first 213 amino acid residues, they are classified into two major types, .alpha. and .beta., based on two variant EGF-like domains which differ in their C-terminal portions. Nevertheless, these EGF-like domains are identical in the spacing of six cysteine residues contained therein. Nagata et al. describe the solution structure of the EGF-like domain of HRG-.alpha. in EMBO. J., 13(15):3517-3523 (1994). Based on an amino acid sequence comparison, Holmes et al. found that between the first and sixth cysteines in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin (AR), 32% identical to TGF-.alpha., and 27% identical to EGF.
The 44 kDa neu differentiation factor (NDF), which is the rat equivalent of human HRG, was first described by Peles et al., Cell, 69:205-216 (1992); and Wen et al., Cell, 69:559-572 (1992). Like the HRG polypeptides, NDF has an immunoglobulin (Ig) homology domain followed by an EGF-like domain and lacks a N-terminal signal peptide. Subsequently, Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994) carried out "exhaustive cloning" to extend the family of NDFs. This work revealed six distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmes et al., the NDFs are classified as either .alpha. or .beta. polypeptides based on the sequences of the EGF-like domains. Isoforms 1 to 4 are characterized on the basis of the variable justamembrane stretch (between the EGF-like domain and transmembrane domain). Also, isoforms a, b and c are described which have variable length cytoplasmic domains. These researchers conclude that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions. See also EP 505 148; and WO 93/22424 concerning NDF. NDF expression has been studied. In particular, Orr-Urtreger PNAS, U.S.A., 90:1867-1871 (1993), using a mouse NDF probe which includes a partial Ig-like domain, found that NDF expression in mouse embryos was in the central and peripheral nervous system [including neuroepithelium that lines the lateral ventricles of the brain, dorsal root ganglia and ventral horn of the spinal cord (albeit weak) as well as intestine, stomach and adrenal cortex].
Falls et al., Cell, 72:801-815 (1993) describe another member of the neuregulin family which they call acetylcholine receptor inducing activity (ARIA) polypeptide. The chicken-derived ARIA polypeptide stimulates synthesis of muscle acetylcholine receptors. See also WO 94/08007. ARIA is a .beta.-type neuregulin and lacks the entire "glyco" spacer (rich in glycosylation sites) present between the Ig-like domain and EGF-like domain of HRG.alpha., and HRG.beta.1-.beta.3.
Marchionni et al., Nature, 362:312-318 (1993) identified several bovine-derived proteins which they call glial growth factors (GGFs). These GGFs share the Ig-like domain and EGF-like domain with the other neuregulin proteins described above, but also have an amino-terminal kringle domain. GGFs generally do not have the complete "glyco" spacer between the Ig-like domain and EGF-like domain. Only one of the GGFs, GGFII, possessed a N-terminal signal peptide. See also WO 92/18627; WO 94/00140; and WO 94/04560 which refer to GGFs and uses thereof. Other polypeptides with mitogenic activity similar to GGFs have been described. For example, Benveniste et al., PNAS, U.S.A., 82:3930-3934 (1985) describe isolation of a glial growth-promoting factor (GGPF) from human T-lymphocyte supernatant which has an apparent molecular weight of 30 kDa on non-reducing SDS-PAGE and 18 kDa on reducing SDS-PAGE. Furthermore, Davis and Stroobant, J. Cell Biol., 110: 1353-1360 (1990) describe various growth factors which are mitogens for rat sciatic nerve Schwann cells in vitro, including GGF, TGF-.beta.1 and TGF-.beta.2.
The various neuregulin domains have been reviewed by Peles and Yarden in Bioessays, 15(12):815-824 (1993). These domains shall be briefly described below.
Inununoglobulin (Ig)-like domain--All of the identified neuregulins for which amino acid sequence data is available possess an Ig-like domain, suggesting this domain has an essential function. See Peles and Yarden, supra.
However, Kuo et al., by screening chick brain cDNA and human cerebellar cDNA libraries, recently identified a novel ARIA splice variant (called nARIA) which lacked the Ig-like domain of the other neuregulin polypeptides. Northern blot analysis localized expression of nARIA in the nervous system with particularly high levels detected in the cerebellum. Kuo et al., Abstract No. 452.18, Soc. Neuroscience Abst., 20:1095 (1994). See also Yang et al., Abstract No. 452.17, Soc. Neuroscience Abst., 20:1095 (1994).
Glycosylated spacer domain--This "glyco" domain, rich in N- and O-linked glycosylation sites, connects the Ig-like domain with the EGF-like domain. A single potential site for glycosaminoglycan attachment is present at the amino terminus of this spacer domain. ARIA and GGF lack 34-amino acid-residues of the glyco spacer, reducing the number of glycosylation sites in these molecules.
EGF-like domain--This region incorporates six cysteine residues and is predicted to fold into a structure having three disulfide-linked loops. As discussed above, neuregulins are classified as either .alpha. or .beta.-type neuregulins based on the sequence of the C-terminal portion of the EGF-like domain. The EGF-like domain alone has been shown to bind with high affinity to the receptor. See Holmes et al., supra and Peles et al., EMBO J., 12: 961-971 (1993). Sequence differences between the .alpha. and .beta. forms apparently do not alter receptor specificity. However, the .beta. form may bind with greater affinity to responsive cells than the .alpha. form.
The C-terminal portion of the EGF-like domain of some neuregulins is flanked by an eight-amino acid stretch. While this stretch of amino acid residues does not appear to affect receptor binding, it is thought to play a role in proteolytic processing of the precursor neuregulin to release the soluble polypeptide. See Peles and Yarden, supra.
Cytoplasmic tail--The transmembrane forms of neuregulins have four different hydrophilic cytoplasmic tails, the largest of which is 415 amino acid residues long. Some of the described neuregulins lack this cytoplasmic tail (e.g. HRG-.beta.3 and GGFII).
Other polypeptides which are considered to be HER2 activators have been described. Lupu et al., Science, 249:1552-1555 (1990) and Lupu et al., Biochemistry, 31:7330-7340 (1992) describe a 30 kDa glycoprotein (termed gp30) with TGF-.alpha.-like properties. Lupu et al. found that this factor stimulated phosphorylation of both the EGFR and p185.sup.HER2. Lupu et al. isolated another growth factor from the conditioned medium of SK-BR-3 cells which they describe as binding to p185.sup.HER2, but not EGFR. This factor, called p75, competed with the anti-HER2 receptor antibody 4D5 for p185.sup.HER2 binding. Lupu et al., Abstract No. 297, Proc. Am. Assoc. Cancer Res., 32 (1991). See also WO 91/18921; WO 92/12174; and WO 93/22339.
There has been another report of a glycoprotein which is able to activate both the EGFR and p185.sup.HER2. This polypeptide had a molecular weight of 35 kDa and was heat stable but sensitive to reduction. See Yarden and Peles, Biochemistry, 30:3543-3550 (1991).
Tarakhovsky et al. describe a 25 kDa polypeptide secreted by activated mouse peritoneal macrophages, which is considered to be a ligand for p185.sup.neu. Tarakhovsky et al., Oncogene, 6(12):2187-2196 (1991). Huang and Huang reported purification of a 25 kDa protein from bovine kidney which they call neu/erbB2 ligand growth factor (NEL-GF). NEL-GFphosphorylated the neu receptor in intact NIH-3T3 cells expressing neu receptor.
Davis et al., Biochem. Biophys. Res. Commun., 179(3):1536-1542 (1991) and Dobashi et al., PNAS, U.S.A., 88:8582-8586 (1991) characterized a protein they call neu protein-specific activating factor (NAF) which had a molecular weight between about 8 to 24 kDa. NAF was said to activate p185.sup.neu but not the EGFR. See also WO 91/15230 to Greene et al. which mentions a 7-14 kDa polypeptide. In a later report, NAF is said to have a molecular weight of 15-17 kDa. Samanta et al., PNAS, U.S.A., 91:1711-1715 (1994).
De Corte et al. recently reported the presence of a 50 kDa protein in conditioned medium of COLO-16 human cancer cells which appeared to activate the HER2 receptor in SK-BR-3 cells. Various biological activities including inducement of fast spreading, fast plasma membrane ruffling, cell shape change, net translocation, stimulation of chemotaxis and growth arrest in SK-BR-3 cells were attributed to this factor. De Corte et al., J. Cell Science, 107: 405-416 (1994).
Diverse biological activities for the various neuregulin polypeptides have been described.
While the heregulin and NDF polypeptides were first identified based on their ability to activate p185.sup.HER2/neu (see Holmes et al., supra), it was discovered that certain ovarian cells expressing neu and neu-transfected fibroblasts did not bind or crosslink to NDF, nor did they respond to NDF to undergo tyrosine phosphorylation [Peles et al., EMBO J., 12:961-971 (1993)]. This indicated another cellular component was necessary for conferring full neuregulin responsiveness. Carraway et al. subsequently demonstrated that .sup.125 I-rHRG.beta.1.sub.177-244 bound to NIH-3T3 fibroblasts stably transfected with bovine erbB3 but not to non-transfected parental cells. Accordingly, they conclude that erbB3 is a receptor for HRG and mediates phosphorylation of intrinsic tyrosine residues as well as phosphorylation of p185.sup.erbB2/neu in cells which express both receptors. Caraway et al., J. Biol. Chem., 269(19):14303-14306 (1994). Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994) found that cells transfected with HER3 alone show low affinities for heregulin, whereas cells transfected with both HER2 and HER3 show higher affinities.
This observation correlates with the "receptor cross-talking" described previously by Kokai et al., Cell, 58:287-292 (1989); Stern et al., EMBO J., 7:995-1001 (1988); and King et al., 4:13-18 (1989). These researchers found that binding of EGF to the EGFR resulted in activation of the EGFR kinase domain and cross-phosphorylation of p185.sup.neu. This is believed to be a result of ligand-induced receptor heterodimerization and the concomitant cross-phosphorylation of the receptors within the heterodimer [Wada et al., Cell, 61:1339-1347 (1990)].
Plowman and his colleagues have similarly studied p185.sup.HER4 /p185.sup.HER2 activation. They expressed p185.sup.HER2 alone, p185.sup.HER4 alone, or the two receptors together in human T lymphocytes and demonstrated that neuregulin is capable of stimulating tyrosine phosphorylation of p185.sup.HER4, but could only stimulate p185.sup.HER2 phosphorylation in cells expressing both receptors. Plowman et al., Nature, 336:473-475 (1993). Thus, neuregulin is the only known example of a member of the EGF growth factor family that can interact with several receptors. Carraway and Cantley, Cell, 78:5-8 (1994).
The biological role of neuregulin has been investigated by several groups. For example, Falls et al., (discussed above) found that ARIA plays a role in myotube differentiation, namely affecting the synthesis and concentration of neurotransmitter receptors in the postsynaptic muscle cells of motor neurons. Corfas and Fischbach demonstrated that ARIA also increases the number of sodium channels in chick muscle. Corfas and Fischbach, J. Neuroscience, 13(5):2118-2125 (1993). It has also been shown that GGFII is mitogenic for subconfluent quiescent human myoblasts and that differentiation of clonal human myoblasts in the continuous presence of GGFII results in greater numbers of myotubes after six days of differentiation (Sklar et al., J. Cell Biochem., Abst. W462, 18D, 540 [1994]).
Peles et al. (1992), supra, and Wen et al. (1992), supra, found that when certain mammary tumor cells (e.g. AU-565 and MDA-MB-453) were exposed to NDF, NDF induced phenotypic differentiation (including morphological changes and synthesis of milk components) and resulted in growth arrest. On the contrary, Holmes et al., supra, found that HRG exerted a mitogenic effect on mammary cell lines (such as SK-BR-3 and MCF-7).
The mitogenic activity of GGFs on Schwann cells has been reported. See, e.g., Brockes et al., J. Biol. Chem., 255(18): 8374-8377 (1980); Lemke and Brockes, J. Neurosci., 4:75-83 (1984); Brockes et al., J. Neuroscience, 4(1):75-83 (1984); Brockes et al., Ann. Neurol., 20(3):317-322 (1986); Brockes, J., Methods in Enzym., 147:217-225 (1987) and Marchionni et al., supra. Shah et al. report that GGF suppresses neuronal differentiation of rat neural crest stem cells but promotes or allows glial differentiation. Shah et al., Cell, 77:349-360 (1994).
Schwann cells constitute important glial cells which provide myelin sheathing around the axons of neurons, thereby forming individual nerve fibers. Thus, it is apparent that Schwann cells play an important role in the development, function and regeneration of peripheral nerves. The implications of this from a therapeutic standpoint have been addressed by Levi et al., J. Neuroscience, 14(3):1309-1319 (1994). Levi et al. discuss the potential for the construction of a cellular prosthesis comprising human Schwann cells which could be transplanted into areas of damaged spinal cord. Accordingly, these researchers outline the need for Schwann cell mitogens which can be used to allow full differentiation of these cells ex vivo. WO 94/00140 describes the use of various factors for stimulating mitogenesis of glial cells (e.g. Schwann cells). Others have demonstrated that heregulin is a potent mitogen for human Schwann cells in vitro. Levi et al., J. Cell Biol., in press.
Pinkas-Kramarski et al. found that NDF seems to be expressed in neurons and glial cells in embryonic and adult rat brain and primary cultures of rat brain cells, and suggested that it may act as a survival and maturation factor for astrocytes (Pinkas-Kramarski et al., PNAS, U.S.A., 91:9387-9391 [1994]). Meyer and Birchmeier, PNAS, U.S.A., 91:1064-1068 (1994) analyzed expression of neuregulin during mouse embryogenesis and in the perinatal animal using in situ hybridization and RNase protection experiments. These authors conclude that, based on expression of this molecule, neuregulin plays a role in vivo as a mesenchymal and neuronal factor. Also, their findings imply that neuregulin functions in the development of epithelia. Similarly, Danilenko et al., Abstract 3101, FASEB, 8(4-5): A535 (1994), found that the interaction of NDF and the HER2 receptor is important in directing epidermal migration and differentiation during wound repair.
It is apparent from the above discussion that neuregulins, similar to other growth factors (e.g. interleukin-6 and amphiregulin), can act as differentiation factors or as mitogens depending on their concentration and on the cellular context.
Accordingly, it is an object of the present invention to identify a novel polypeptide activator of the HER2 receptor and/or a glial cell mitogen for diagnostic, ex vivo, and therapeutic uses.
It is yet another object to provide nucleic acid encoding such polypeptide and to use this nucleic acid to produce the polypeptide in recombinant cell culture.
It is a still further object to provide derivatives and modified forms of such polypeptide, including amino acid sequence variants and covalent derivatives thereof.
It is an additional object to prepare immunogens for raising antibodies against such polypeptides, as well as to obtain antibodies capable of binding them.
These and other objects of the invention will be apparent to the ordinarily skilled artisan upon consideration of the specification as a whole.